Absolute Protein Quantification Using AQUA-Calibrated 2D-PAGE
Absolute protein quantification for the analysis of proteome dynamics is more and more required by the scientific community. Therefore a number of methods have recently been reported that aim at determining concentrations of single proteins in complex samples, all of them having their advantages and limitations. However, for all of these methods an accurate and protein unspecific determination of the total protein amount in a given sample is urgently needed. Here a ninhydrin-based assay established to reach this goal is described. Moreover, an optimized protocol for protein digestion is an inevitable prerequisite for all mass spectrometry-based approaches aiming at absolute protein quantification. In this chapter, various aspects are described which have to be considered during validation of a suitable digestion method and a detailed protocol is presented that can be applied to the digestion of soluble proteins originated from microbes.
In order to provide an absolute protein quantification workflow applicable for small scale and large scale approaches, a step-by-step guide is provided for the so-called AQUA-strategy (AQUA = absolute quantification), including selection of suited standard peptides, the development of optimized MS methods and the determination of absolute protein concentration using stable isotope dilution and selected reaction monitoring (SID-SRM). Subsequently, a workflow is introduced that combines targeted mass spectrometry and two-dimensional polyacrylamide gel electrophoresis for the large-scale determination of absolute protein amounts.
Key wordsProteomics Absolute protein quantification Protein Digestion AQUA Stable isotope dilution Selected reaction monitoring 2D-PAGE
This work was supported by the German Research Foundation Grant SFB/TR34.
- 4.Muntel J, Fromion V, Goelzer A, Maaβ S, Mäder U, Büttner K, Hecker M, Becher D (2014) Comprehensive absolute quantification of the cytosolic proteome of Bacillus subtilis by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE). Mol Cell Proteomics 13(4):1008–1019CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275Google Scholar
- 15.Maass S, Sievers S, Zühlke D, Kuzinski J, Sappa PK, Muntel J, Hessling B, Bernhardt J, Sietmann R, Völker U, Hecker M, Becher D (2011) Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal Chem 83(7):2677–2684CrossRefPubMedGoogle Scholar
- 16.Piehowski PD, Petyuk VA, Orton DJ, Xie F, Moore RJ, Ramirez-Restrepo M, Engel A, Lieberman AP, Albin RL, Camp DG, Smith RD, Myers AJ (2013) Sources of technical variability in quantitative LC-MS proteomics: human brain tissue sample analysis. J Proteome Res 12(5):2128–2137CrossRefPubMedPubMedCentralGoogle Scholar
- 27.Desiere F, Deutsch EW, Nesvizhskii AI, Mallick P, King NL, Eng JK, Aderem A, Boyle R, Brunner E, Donohoe S, Fausto N, Hafen E, Hood L, Katze MG, Kennedy KA, Kregenow F, Lee H, Lin B, Martin D, Ranish JA, Rawlings DJ, Samelson LE, Shiio Y, Watts JD, Wollscheid B, Wright ME, Yan W, Yang L, Yi EC, Zhang H, Aebersold R (2005) Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry. Genome Biol 6(1):R9CrossRefPubMedGoogle Scholar
- 40.Kuntumalla S, Braisted JC, Huang S-T, Parmar PP, Clark DJ, Alami H, Zhang Q, Donohue-Rolfe A, Tzipori S, Fleischmann RD, Peterson SN, Pieper R (2009) Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Leptospira interrogans proteome. Proteome Sci 7:22CrossRefPubMedPubMedCentralGoogle Scholar
- 44.Kraut A, Marcellin M, Adrait A, Kuhn L, Louwagie M, Kieffer-Jaquinod S, Lebert D, Masselon CD, Dupuis A, Bruley C, Jaquinod M, Garin J, Gallagher-Gambarelli M (2009) Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. J Proteome Res 8(7):3778–3785CrossRefPubMedGoogle Scholar